EXPERIMENTAL CELL RESEARCH 241, 66–75 (1998)ARTICLE NO. EX984005

S- and G2-phase Cell Cycle Arrests and Apoptosis Inducedby Ganciclovir in Murine Melanoma Cells Transduced

with Herpes Simplex Virus Thymidine Kinase

Sung-Jen Wei,*,† Yee Chao,‡ Yi-Mei Hung,§ Wen-chang Lin,† Den-Mei Yang,§ Yung-Luen Shih,§Lan-Yang Ch’ang,† Jacqueline Whang-Peng,†,§ and Wen K. Yang†,§,1

*Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 90048, Taiwan; †Institute of Biomedical Science, AcademiaSinica, Taipei 11529, Taiwan; §Cooperative Clinical Cancer Research Laboratory, Veterans General Hospital-Taipei, Cancer ResearchDivision, National Health Research Institutes, Taipei 11217, Taiwan; and ‡Division of Gastroenterology, Veterans General Hospital-

Taipei and Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan 11217, Republic of China

highly toxic metabolites [1]. One such ‘‘suicide gene’’Mechanism of cell killing by transfer of Herpes sim- is the thymidine kinase gene of Herpes simplex virus

plex virus type-1 thymidine kinase (HSVtk) and subse- (HSVtk). Unlike mammalian cell thymidine kinase,quent ganciclovir (GCV) treatment was examined in HSVtk is able to efficiently catalyze the phosphoryla-B16F10 murine melanoma model. While parental tion of relatively nontoxic anti-herpetic drug gan-B16F10 melanoma cells were resistant to GCV at 100 ciclovir (GCV), a guanosine analogue, into monophos-mM or higher, HSVtk-transduced B16F10 melanoma phate form and, with further catalysis by cellularcell clones became susceptible to GCV with IC50 of 0.1 nucleotide kinases, into highly toxic ganciclovir tri-to 0.3 mM. By means of various parameters including phosphate [2]. Presumably, the utilization of this nu-characteristic morphological changes, in situ DNA cleoside analogue triphosphate as a substrate for DNAend-labeling, DNA ladder pattern, flow cytometric de- replication can cause premature chain termination andtection of sub-G1 DNA content, and annexin V binding lead to cell death [3]. Thus, transfer of HSVtk geneof inverted cell surface phosphatidylserine, apoptosis into tumor cells followed by treatment with GCV wouldwas shown to be associated with the cell killing of gan- provide a novel strategy for cancer therapy [4]. Thisciclovir on HSVtk-transduced melanoma B16F10 cells.

strategy has been investigated in several different ma-Kinetic analysis showed that the signs of apoptosislignant tumor cell lines, including lymphoma [5], hepa-were observed not until 60 h of continued GCV treat-tocellular carcinoma [6–8], glioma [9–12], ovarian car-ment and preceded first by a rise in p53 protein levelcinoma [13], breast carcinoma [14], fibrosarcoma [4],in 12 h and then by S-phase/G2-phase cell cycle arrestadenocarcinoma [15], and melanoma [16, 17]. However,associated with corresponding increases in the levelmolecular mechanism of tumor cell killing associatedof cyclin B1 protein but no apparent change in proteinwith the HSVtk/ganciclovir therapy remain to be fullylevel of Bax or Cdc2. These results suggest thatelucidated [4, 18]. This is important also for under-apoptosis occurred as a result of ganciclovir-inducedstanding of the ‘‘bystander effect’’ [19], namely thatcell cycle arrests rather than direct chemical effect

on HSVtk-transduced B16F10 melanoma cells. q 1998 GCV treatment causes cytotoxicity and cell death notAcademic Press only to the HSVtk-transduced tumor cells but also to

Key Words: apoptosis; bystander effect; HSVtk; GCV; nearby tumor cells, which are not transduced withcell cycle; melanoma cells. HSVtk.

Programmed cell death (PCD), or apoptosis, has beensuggested as a cytocidal mechanism of the HSVtk-ex-pressing cells when exposed to GCV. In a study of hu-INTRODUCTIONman colon cancer cells, Freeman et al. [20] demon-strated that the bystander effect, i.e., cytocidal effectAn approach to gene therapy of cancer is based on theof GCV on the neighboring HSVtk-negative HCT cells,transfer of nonmammalian genes that encode enzymes

capable of selectively converting nontoxic prodrug into also appeared to be due to apoptosis, as suggested byflow cytometric and electron microscopic observationsthat apoptotic vesicles generated from the dying gene-

1 To whom correspondence and reprint requests should be ad- modified cells were phagocytized by nearby, nontrans-dressed at Cooperative Clinical Cancer Research Laboratory, Veter-duced tumor cells. Similar results were also obtainedans General Hospital-Taipei, NO. 201, Sec. 2, Shih-Pai Road, Taipei

11217, Taiwan, R.O.C. Fax: 886-2-873-3664. with human U-87 glioma cells by Colombo et al. [21]

660014-4827/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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67HSVtk/GCV-INDUCED APOPTOSIS

Measurement of ganciclovir-induced cytotoxicity. The cytocidaland with rat C6 glioma cells by Samejima et al. [22].effect of GCV was determined by colorimetric quantitation of viableConversely, Kaneko and Tsukamoto [23] reported thatcells [30]. Then 2 1 104 of the parental B16F10, B16F10/TK-C15,GCV-induced death of HSVtk-expressing hepatocellu- or -C19 cells were plated, respectively, on the 96-well plates and

lar carcinoma cells was mediated through a nonapo- incubated overnight. Cells were treated on day 0 with GCV (Cyto-vene; Syntex Inc., Palo Alto, CA, U.S.A.) at a concentration of 0, 0.01,ptotic pathway because they did not observe the ap-0.1, 0.5, 1.0, 10, and 100 mM. Medium was changed on day 2 withpearence of DNA ladders. Whether it is due to differ-the corresponding fresh GCV medium. Ninty-two hours later, 50 mlences in the cancer cell target or in the experimentalof 0.5% MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumparameters used for assessing the apoptotic changes bromide] (Sigma) in phosphate-buffered saline (PBS) was added, in-

remains to be determined. cubated for 4 h, removed, and replaced with 100 ml of dimethylsulfox-Recently, the HSVtk/ganciclovir therapeutic ap- ide (DMSO) (Merck) to extract formazan. The color reaction was

measured using an ELISA reader (Bio-Rad) at 570 nm. The cellproach has been employed successfully in murine mela-viability was also assessed by trypan blue exclusion staining.noma animal model, especially by using a tissue- or

Detection of HSVtk gene. To detect the HSVtk gene in transducedtumor-specific promoter, e.g., tyrosinase promoter incell lines, hot start polymerase chain reaction (HS-PCR) was em-melanoma, to regulate and restrict expression of theployed as described previously [31]. Then 5 1 106 of MN48-tk0,

therapeutic gene in the target tumor cells [16, 17]. It B16F10, B16F10/TK-C15, and -C19 cells were plated, respectively,is of interest to know whether the HSVtk/GCV-induced in 10-cm dishes and cultured for 48 h. Total genomic DNA was iso-

lated for detection of the HSVtk gene. HSVtk gene was synthesizedcell death and the bystander effect are mediatedby HS-PCR and analyzed on 0.8% agarose gel. Gels were blotted ontothrough the apoptotic mechanism on melanoma cells.nylon membranes (Amersham, Arlington Heights, IL, U.S.A.), andIn the present study, we have analyzed the GCV-membranes were then hybridized with a high-specific-activity 32P-induced cell death in transduced cell clones of HSVtk- labeled TK DNA probes in 5 1 SSC (1 1 SSCÅ150 mM NaCl and

expressing B16F10 murine melanoma both in vitro and 15 mM sodium citrate), 1% SDS, 1 1 Denhardt’s solution, and 100mg/ml salmon sperm DNA at 657C for 12 h. The DNA probe wasin vivo by using a variety of different methods to iden-synthesized by using TK1 (5*-TGC AGC GAC CCG CTT AAC AGCtify apoptotic cells, including fluorescein-annexin VGT-3 *) and TK2 (5*-CAT AGA TCT GGA TCC TTC CGG TAT TGTbinding assay [24]. Our data indicate that the apoptosisCT-3 *) primers (Quality Systems Inc., U.S.A.). The filters weremechanism is mainly associated with GCV-induced cell washed in 0.05 1 SSC and 0.1% SDS at 657C for 45 min and exposed

cycle S/G2 phase arrest and elevation of p53 and cyclin to X-ray films for 1 min.B1 protein levels. Flow cytometric analysis. Flow cytofluorometry was employed for

the analysis of cellular DNA contents [32] and quantitation of cellsundergoing apoptosis [33]. B16F10, B16F10/TK-C19 cells or theirMATERIALS AND METHODS1:1 mixture were plated in 10-cm dishes at a density of 1 1 106 cells.Twenty-four hours later, the cells were treated with or without 3 mM

Mice and cell lines. Six- to eight-week-old female syngeneic GCV in growth medium for 24, 48, 60, and 72 h. To evaluate theC57BL/6 mice were obtained from central animal facility of the Tai- cellular DNA content, 51 105 of cells were washed once with PBS andwan University Medical Center. All mice were maintained and han- fixed in 80% ethanol at 0207C for at least 1 h. After centrifugation atdled according to the U.S. NIH guideline. NIH3T3-derived MN48- 800 rpm for 5 min at 47C, cell pellet was permeabilized by suspensiontk0cells (from R.H. Bessin, US-NCI) and packaging cell line PA317

in 0.5 ml of 0.1% Triton X-100 for 30 min, washed, and then mixed[25] or f-2 [26] were maintained in minimal essential mediumwith 1 ml of 50 mg/ml propidium iodide (PI) plus 0.5% (w/v) of RNase(MEM) (Sigma Chemical Co., St. Louis, MO, U.S.A.) supplementedA in the dark. After 10 to 30 min, DNA content was analyzed bywith 5% FBS (HyClone, Logan, UT, U.S.A.) and 5% BCS (HyClone),flow cytofluorometry (FACStar plus; Becton–Dickinson). To quanti-2 mM of L-glutamine, 100 U/ml of penicillin, and 100 mg/ml of strepto-tatively measure the percentage of cells undergoing apoptosis, 100mycin sulfate (all from Life Technologies Inc., Grand Island, NY,ml of the cells (1 1 105) suspended in 1 1 binding buffer (Hepes-U.S.A.). B16F10 melanoma cells, given kindly by Dr. I. J. Fidler (M.buffered saline solution supplemented with 25 mM CaCl2) wereD. Anderson Cancer Center, University of Texas, Houston, TX,transfered to a 5 ml culture tube. Ten microliters of 10 mg/ml FITC-U.S.A.), were grown in MEM with 10% FBS. All cell lines were incu-conjugated annexin V and 10 ml of 50 mg/ml PI reagents (Apoptosisbated at 377C in a humidified atmosphere of 5% CO2/95% air. CellDetection kit, R&D) were added and then incubated for 15 min atlines used in this study were free of mycoplasma infection.room temperature in the dark. Flow cytofluorometric analysis was

Transduction of murine melanoma cells. A recombinant retrovi- performed within 1 h after the staining procedure.ral vector was constructed with proviral DNA frame of pWN-B5 [27]

DNA ladder assay. Cellular DNA fragments were isolated by thethat contains HSV-1-tk [28] with its promoter and neoR gene withmethod described by Hermann et al. [34] with minor modifications.simian virus 40 early promoter. Packaging of the vector was per-Various times after GCV treatment, adherent and nonadherent cellsformed according to the published procedures [29] by employing c-were pooled and lyzed in 50 mM Tris–HCl (pH 7.5), 20 mM EDTA,2 and PA317 sequentially to produce high titer HSV-tk/carrying am-and 1% NP-40. After centrifugation for 1 min at 3000 rpm, the super-photropic retroviral virion. B16F10 (5 1 106) melanoma cells werenatant was collected, and the extraction of pellets repeated twice.pretreated for 30 min with serum-free medium containing 20–25 mg/Pooled supernatants were brought to 1% SDS, digested with DNase-ml of DEAE-dextran. The cells were then incubated for 4 h in 3 mlfree RNase A (final concentration 0.2 mg/ml) at 507C for 1 h, and thenof the amphotropic virion-containing medium plus 4 ml of serum-with proteinase K (final concentration 2.5 mg/ml) at 377C overnight.free medium. Subsequently, the cells were exposed to the growthAfter the digestion, DNA was isolated by phenol/chloroform extrac-medium containing 375 mg/ml G418 for 2 weeks. Individual B16F10/tion and ethanol precipitation and finally dissolved in 10 mM Tris–TK cell clones were isolated by G418 resistance. Two transducedHCl/1 mM EDTA solution (pH 7.6). The DNA preparations werecell clones, B16F10/TK-C15 and B16F10/TK-C19, were selected forseparated in 1% agarose gel by electrophoresis and stained withfurther studies on the basis of their high sensitivity to the cytocidal

effect of GCV. ethidium bromide.

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68 WEI ET AL.

In situ DNA fragment end-labeling. Detection of DNA fragmenta-tion in situ was performed according to a published procedure [35].B16F10, B16F10/TK-C15, and -C19 were plated at 2 1 105 per wellthat contained a 60-mm coverslip for 24 h and then treated with 3mM GCV. After 24, 48, and 60 h of treatment, the cells on the coverslipwere then fixed in 4% paraformaldehyde/sodium phosphate buffer(pH 7.4) for at least 15 min at 47C. After rehydration and proteinaseK digestion, cells were incubated in 60 ml of labeling reaction mixtureconsisting of 50 mM Tris (pH 8.0), 50 mM NaCl, 10 mM MgCl2,1 unit Escherichia coli DNA polymerase I large fragment (Klenowenzyme), 0.2 nmol unlabeled dATP, dCTP, and dGTP, and 0.2 nmolbiotin-labeled dUTP at 377C for 1.5 h. After inactivation of endoge-nous peroxidases with 3% H2O2, the coverslip specimen was incu-bated with conjugation solution of streptavidin-horseradish peroxi-dase (SA-HRP) in blocking buffer, covered with freshly prepared DABsolution in H2O2/Urea tap water, counterstained with methyl greensolution, and finally mounted with permount for observation by lightmicroscope (Nikon, Japan).

Western blot analysis. Trypsinized cells were washed with ice-cold PBS once and then solubilized in 50 mM Tris–HCl (pH 7.5), 4mM EDTA, 2 mM EGTA, 10 mM DTT, and 1 mM PMSF. Lysateswere sonicated on ice and centrifuged at 100,000g for 1 h at 47C. Thepellet and supernatant were stored at0807C. Protein concentrationswere determined by protein assay (Bio-Rad). Supernatant proteinswere separated on 10% SDS/polyacrylamide gels and transferredto polyvinylidene difluoride membrane. The membrane blots wereincubated with 0.5 mg/ml of anti-p53 Ab-1 (Oncogene Science, MA,U.S.A.), anti-Bax, or anti-cyclin B1 (Santa Cruz, CA, U.S.A.), andfollowed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG at 1:3000 dilution (Amersham). Protein bands of P53,Bax, or cyclin B1 were detected by enhanced chemiluminescence(ECL) (Amersham). After fluorography, membranes were stripped

FIG. 1. Correlation of in vitro GCV sensitivity and HSVtk geneand then rehybridized with anti-Cdc2 protein to serve as an internalstatus. [A] Parental B16F10, B16F10/TK-C15, or -C19 cells, platedcontrol.at 2 1 104 per well in 96-well multiplates, were exposed to the GCVIn vivo animal studies. To evaluate the apoptotic effects of GCVat concentrations ranging from 0 to 100 mM. After 92 h, MTT wastreatment on the HSVtk-expressing B16F10 cells in vivo, 106 of pa-added and incubated for a further 4 h. Formazan was extracted withrental HSVtk-negative B16F10, HSVtk-positive B16F10/TK-C19DMSO and measured by ELISA reader at 570 nm. Data representcells were inoculated subcutaneously into the female syngeneicthe mean and standard error of mean of three separate experimentsC57BL/6 mice at the left and the right flank, respectively. Sevencarried out in quadruplicate. [B] Following the hot start PCR, pres-days later, mice were treated by intraperitoneal injection of 150 mgence of the integrated HSVtk gene in transduced cell lines was de-GCV per kg body weight twice a day for 5 consecutive days. Followingtected by hybridization with specific DNA probe. Only the HSVtk-the GCV treatment, the tumor mass was excised and fixed in 4%transduced cell lines, B16F10/TK-C15 (lane 7) and -C19 (lane 6), andparaformaldehyde in sodium phosphate buffer (pH 7.4) for at leastpositive control plasmid pKMKO (lane 2) showed a band of correct4 h at 47C and then processed for paraffin embedding. The specimensize (Ç1.3 kb). MN48-tk0 cells (lane 4) and ddH2O (lane 3) servedwas cut into 4- to 6-mm sections and mounted on glass slides. Sectionsas negative control. Lanes 1 and 5 represent 1-kb marker and HSVtk-were histochemically stained by in situ DNA fragment end-labelingnegative parental B16F10 cells, respectively.procedure as described above.

tive to the drug GCV at concentrations as low as 0.5 mM,RESULTSalthough a small percentage of gene-modified cells wereresistant to GCV even at the highest concentration (100Selective Toxicity of GCV in HSVtk-ExpressingmM). The concentration causing 50% inhibition of theTumor CellsHSVtk-transduced B16F10 cells was similar (IC50: 0.1 to

Genetically modified HSVtk-positive B16F10 tumor 0.3 mM) for the C15 and C19 clones. All HSVtk-negativecell clones were obtained by transduction of B16F10 cells parental B16F10 cells remained viable in GCV even atwith amphotropic retroviral vector carrying HSVtk and 100 mM. The persence of the HSVtk gene (Fig. 1B) in theneoR genes and selected in MEM medium containing neo- transduced tumor cell clones was confirmed by PCR ofmycin analogue G418. The HSVtk-negative parental mel- cellular DNA with the detection of a predicted Ç1.3 kbanoma cells, B16F10, and two HSVtk-positive cell clones, specific DNA fragment.B16F10/TK-C15 and -C19, were grown, respectively, in

HSVtk/GCV-Induced Programmed Cell DeathMEM medium containing 0, 0.01, 0.1, 0.5, 1.0, 10, andin Melanoma Cells100 mM of GCV and the viable cells were measured by

MTT assay after a 96-h exposure. As shown in Fig. 1A, Morphological changes of HSVtk-positive B16F10/TK-C19 and nontransduced parental B16F10 cells ex-the tumor cells which contain the HSVtk gene were sensi-

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69HSVtk/GCV-INDUCED APOPTOSIS

FIG. 4. In vitro quantitation of HSVtk/GCV-induced apoptosis.The B16F10 or B16F10/TK-C19 cells were treated with 3 mM GCV for60 h [A and B] and 72 h [C and D]. The treated cells were trypsinized,washed, and suspended in 1 1 binding buffer (10 mM Hepes buffersupplemented with 25 mM CaCl2) in a 5-ml tube. Cells were stainedwith 1 mg/ml FITC-conjugated annexin V and 5 mg/ml PI reagentsfor 15 min before analysis by flow cytometry. The percentages of cellsat an early stage of apoptosis, as measured by single annexin Vfluorescence, and the fraction of cells at an advanced stage ofapoptosis, as measured by dual annexin V/PI fluorescence.

posed to growth medium containing 3 mM of GCV wereexamined at various times by light microscopy (Fig. 2).At 48 h, B16F10/TK-C19 cells became larger in size(Fig. 2E) than untreated control cells. After 60 h ofGCV exposure, characteristics of apoptosis or pro-grammed cell death (PCD), namely, membrane bleb-bing, cell detachment, cell shrinkage, vesicle formation,compaction and margination of nuclear chromatin, andconvolution of nuclei were conspicuous in B16F10/TK-C19 cells (Figs. 2G and 2I), while no such effects wereobserved in the control HSVtk-negative parentalB16F10 cells (Fig. 2A) nor in HSVtk-positive cells with-out GCV treatment (Fig. 2C). The mixed HSVtk-posi-tive and -negative cell culture exposed to 3 mM GCVshowed the similar frequent features of apoptosis, ap-parently affecting both the transduced and nontrans-duced bystanding cells. The occurrence of PCD was fur-ther verified by in situ DNA fragment end-labeling ofapoptotic nuclei. As expected, HSVtk-negative parentalB16F10 cells treated with 3 mM GCV (Fig. 2B) andFIG. 2. HSVtk/GCV-induced alterations of melanoma cells ob-untreated HSVtk-positive B16F10/TK-C19 cells (Fig.served in cell cultures [A, C, E, G, I] and by in situ DNA fragment

end-labeling analysis [B, D, F, H, J]. HSVtk-negative B16F10 cells 2D) exhibited intact nuclear structure and minimalwere exposed to 3 mM GCV for 60 h [A, B], while B16F10/TK-C19 end-labeling, whereas GCV-treated B16F10/TK-C19cells were untreated [C, D] or treated with 3 mM GCV for 48 h [E, F], cultures examined at 48, 60, and 72 h (Figs. 2F, 2H, and60 h [G, H], and 72 h [I, J]. Apoptotic nuclei revealed by nonisotopic

2J) contained increasing numbers of cells with apop-histochemical DNA end-labeling are indicated by arrows. Photo-graphs were taken at 2001 magnification. totic nuclei. By 72–96 h of GCV treatment, nearly all

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70 WEI ET AL.

FIG. 7. HSVtk/GCV-induced apoptosis in subcutaneous tumor of B16F10 melanoma in vivo. Tumor formation in 6- to 8-week-oldsyngeneic female C57BL/6 mice, GCV treatment, tumor specimen processing, and in situ DNA fragment end-labeling procedures weredescribed under Materials and Methods. [A], [C], [E] represent the untreated parental B16F10 tumor, while [B], [D], [F] are the treatedB16F10/TK-C19 tumor. [A] and [B] are stained by hematoxylin and eosin. [C], [D] and [E], [F] are DNA fragment end-labeling of tissuesections at low (401) and high (10001) magnification, respectively. Nuclei of apoptotic cells indicated by an arrow were relatively largerin size.

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71HSVtk/GCV-INDUCED APOPTOSIS

FIG. 3. Internucleosomal DNA cleavage caused by HSVtk/GCV. [A] HSVtk-negative B16F10 and HSVtk-positive B16F10/TK-C19 cells wereuntreated or treated with 3 mM GCV for 0, 24, 48, 60 and 72 h. [B] HSVtk-positive B16F10/TK-C15 cells were untreated or treated with 3 mMGCV for 60 and 72 h. Cells were harvested by trypsinization and DNA was extracted from the cells lyzed in 50 mM Tris–HCl (pH 7.5), 20 mMEDTA, and 1% NP-40. Equal amounts of the total extracted cellular DNA were separated by electrophoresis in 1% agarose gel and visualized bystaining with 0.5 mg/ml ethidium bromide.

cells in the B16F10/TK-C19 and the mixed cultures double fluorescence, presumably due to cell damagesoccurred during experimental procedures (Figs. 4A andcontained an apoptotic nucleus (data not shown).4C). The mixed cell cultures treated with GCV for 72

Induction of Chromosomal DNA Fragmentation and h also contained 12.5% of early stage apoptotic cellsCell Surface Phosphatidylserine Inversion and 60.0% of advanced-stage apoptotic cells (data not

shown).Appearance of oligonucleosomal DNA fragments or

laddering of the cellular DNA has been commonly used Kinetics of Cell Cycle Effects and Related Proteinas an indication of apoptosis. The DNA ladder pattern Changeswas clearly observed in DNA preparations from HSVtk-expressing B16F10/TK-C19 cells treated with 3 mM Flow cytometric analysis was performed to distin-

guish populations of cells at different phases of cycleGCV for 60 or 72 h, although it was not apparent follow-ing 24 or 48 h of exposure (Fig. 3A). As the negative and also apoptotic cells that contain sub-G1 DNA quan-

tity. In regular culture medium without GCV, thecontrol, cellular DNA preparations from parentalB16F10 with or without GCV treatment or from un- HSVtk-transduced B16F10 cell clones and the non-

transduced B16F10 melanoma cells were similar intreated B16F10/TK-C19 cells did not display the pat-tern of DNA laddering. Similar results were obtained containing 52.7% of G1/G0 cells, 37.6% of S-phase cells,

and 7.7% of G2/M-phase cells, with a doubling time offrom B16F10/TK-C15 cell clone treated with GCV for60 or 72 h (Fig. 3B). To further quantitatively deter- about 24 h. Addition of 3 mM GCV in the growth me-

dium, while not affecting the proliferation of nontrans-mine the percentage of cells undergoing apoptosis inthe GCV-exposed B16F10/TK-C19 cells, annexin V duced melanoma cells, inhibited the growth of HSVtk-

transduced B16F10 cells and caused a gradual disap-binding and PI permeability were simultaneously mea-sured (Fig. 4). Sixty hours after being exposed to 3 mM pearance of G1-phase cells and corresponding increase

and accumulation of S-phase cells and G2-phase cellsGCV, 35.6% of the B16F10/TK-C19 cells exhibited earlysigns of apoptosis, i.e., with annexin V binding fluores- in 48 h (Fig. 5). Apoptotic cells were not apparent at

48 h, but became marked at 60 h and prominent at 72cence without PI staining, while 26.3% were in ad-vanced-stage apoptosis, showing annexin V/PI double h. These kinetic results clearly indicated that retarda-

tion of S-phase progression and eventual G2-phase ar-fluorescence (Fig. 4B). At 72 h, cells in early stageapoptosis showed a decrease to 14.9%, while those in rest occurred prior to the emergence of apoptosis in

HSVtk-transduced B16F10 melanoma treated withadvanced-stage apoptosis were raised to 64.0% (Fig.4D). In contrast, only 8% of the HSVtk-negative paren- GCV for 60–72 h (Table 1).

Kinetic series of extracts were prepared from HSVtk-tal B16F10 cells treated with GCV for 72 h showed

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72 WEI ET AL.

TABLE 1

Kinetic Changes in Percentage Distribution of Cell CyclePhases and Apoptosis in B16F10/TK-C19 Cells at VariousTimes of GCV Treatment

Phases

Sub-G1 apoptotic G1 S G2/MHours (%) (%) (%) (%)

0 2.1 52.7 37.5 7.724 2.8 22.6 42.2 32.448 6.6 8.2 51.7 33.560 25.7 5.9 52.6 15.872 54.9 2.4 34.2 8.5

Note. Cells were treated with 3 mM GCV for 0, 24, 48, 60, and 72h. The distribution of cells in G1, S, G2/M phases of cell cycle or insub-G1 apoptotic peak were quantitated by flow cytometric analysis.

tein level of B16F10 melanoma cells, either with orwithout GCV exposure, was below detectable level;whereas in both HSVtk-transduced B16F10 cell clonestreated with GCV p53 rapidly became detectable in 12h, even earlier, and remained at high levels throughoutthe treatment, which was consistent with the presenceof HSVtk/GCV-induced DNA damages in the cell (Fig.6A). Also, in HSVtk-transduced B16F10 cells, GCV ex-posure caused an increase of cyclin B1 protein level in24 h, reaching maximal levels at 48–54 h (Fig. 6D),although the Cdc2 protein level remained constantwith or without HSVtk/GCV treatment (Fig. 6C). No

FIG. 5. Flow cytometric analysis of the cellular DNA content inGCV-exposed HSVtk-positive tumor cells. The B16F10/TK-C19 cellswere treated with 3 mM GCV for 0, 24, 48, 60 and 72 h and thenexamined by flow cytometry as described under Materials and Methods.The sub-G1 peaks were indicated by arrows and similar results wereobtained in two independent experiments, one of which was shown.

FIG. 6. Effect of GCV treatment on apoptosis and cell cycle-re-lated cellular protein levels. B16F10 and B16F10/TK-C19 cells weretreated with 3 mM GCV for 0, 12, 24, 30, 36, 48, 54, and 60 h. Determi-transduced and nontransduced B16F10 cells at variousnation of p53 [A], Bax [B], Cdc2 [C and D], and cyclin B1 proteinstimes after GCV treatment and examined by Western [D] was performed by protein gel blot analysis as described under

blot analysis for p53, Bax, Cdc2, and cyclin B1 proteins Materials and Methods. Cdc2 was not affected by HSVtk/GCV treat-ment and used as an internal control.with respective specific antibodies (Fig. 6). The p53 pro-

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73HSVtk/GCV-INDUCED APOPTOSIS

change in the Bax (Fig. 6B) and Bcl-2 (data not shown) melanoma in the animal treated by intraperitoneal in-jections of ganciclovir.protein levels was observed, suggesting that HSVtk/

GCV-induced apoptosis in melanoma model was not Our results are consistent with previous studieswhich applied HSVtk/ganciclovir strategy and observedmediated through Bax/Bcl-2 pathway.electrophoretic ‘‘DNA ladder’’ pattern in glioma [36].However, in a similar study on HSVtk-expressing XCAnimal Study of HSVtk/GCV-Induced Apoptosisrat hepatoma cells, Kaneko and Tsukamoto [23] ob-in Vivoserved no ‘‘DNA ladder’’ pattern and concluded that

The above in vitro experiments indicated that GCV- apoptosis was not involved in the ganciclovir cytotoxic-induced cytotoxicity in HSVtk-transduced B16F10 mel- ity of this tumor. We also found that ganciclovir treat-anoma cells was mediated through the apoptotic pro- ment could induce internucleosomal DNA fragmenta-cess. To further determine whether apoptotic cell death tion and other features of apoptosis in mouse 1 MEAoccurred in vivo, three groups of C57BL/6 syngeneic 7R.1 hepatoma cells with HSVtk gene, except that rela-mice were inoculated subcutaneously with B16F10, tively more (and yet still a very small fraction) of theB16F10/TK-C19 and 1:1 mixture of B16F10 and transduced hepatoma cells could survive and resumeB16F10/TK-C19 cells. At day 7 after inoculation, mice cell growth after discontinuation of ganciclovir treat-were treated intraperitoneally with 150 mg GCV per ment, suggesting relative resistance of these hepatomakg body weight twice daily for 5 consecutive days. At cells (our unpublished observation). Thus, the thera-day 12, tumor mass was excised and examined by in peutic efficacy of the HSVtk/ganciclovir strategy andsitu DNA fragment end-labeling assay. We found that the apoptotic responses may differ among various tu-GCV administration could effectively cause HSVtk- mors, being more achievable in melanoma than in hep-transduced B16F10/TK-C19 tumors and the cell mix- atoma. Recently, employing pulse-field gel electropho-ture tumors to regress similarily (Fig. 7B) with the resis, several studies have reported that apoptosis canappearance of obvious DNA fragmentation in the nu- proceed with higher order of chromosomal DNA cleav-cleus (Figs. 7D and 7F). In contrast, tumors of the age to produce rosettes and loops, without further deg-HSVtk-negative parental B16F10 cells treated identi- radation into oligonucleosomal DNA fragments [37]. Itcally with GCV continued to grow rapidly (Fig. 7A) with remains to be determined if ganciclovir-induced cellintact nuclear structure (Figs. 7C and 7E). death in HSVtk-transduced hepatoma cells might in-

volve an apoptosis mechanism without internucleoso-DISCUSSION mal DNA cleavage.

In a previous study on ‘‘bystander effect’’ of theHSVtk/ganciclovir strategy in mixed cell cultures, S-In this study, mouse melanoma B16F10 cells were

employed to investigate the cell death mechanism asso- phase arrest of the cell cycle was found in HSVtk-posi-tive PA317 cells as well as in coculturing HSVtk-nega-ciated with the HSVtk/ganciclovir or ‘‘suicidal’’ enzyme/

prodrug strategy of gene therapy against cancer. Our tive C6CX43 glioma cells [38]. Another study reportedthat ganciclovir caused G2-phase arrest in HSVtk-posi-results showed that the melanoma cells transduced

with HSVtk gene indeed became susceptible to gan- tive c-2 cells, although not as obvious in HSVtk-posi-tive XC hepatoma cells [23]. In the present study, ki-ciclovir at concentrations which were nontoxic to the

parental melanoma cells and that the effect of gan- netic analysis revealed that, following ganciclovirtreatment, HSVtk-expressing B16F10 melanoma cellsciclovir was manifested by apoptosis or ‘‘programmed

cell death’’ in the HSVtk-transduced cells. The apop- first exhibited S-phase delay, then G2-phase arrest,and finally apoptosis (Fig. 5). Ganciclovir triphosphate,tosis was evident by various criteria including the ob-

servation of characteristic changes of cell morphology the major end-product of ganciclovir in HSVtk-trans-duced cells, would inhibit cellular DNA polymerasessuch as membrane blebbing, chromatin condensation

and vesicle formation (Fig. 2), appearance of sub-G1 and also would yield erroneous ganciclovir nucleotideincorporation into DNA, resulting in S-phase delay asDNA content or ‘‘apoptotic peak’’ in flow cytometric

analysis (Fig. 5), ‘‘DNA ladders’’ patterns in agarose well as G2-phase arrest by the activation of 3 * exo-nuclease and postreplicative endonuclease repairelectrophoresis, indicating internucleosomal cleavages

of cellular DNA (Fig. 3), histochemical demonstration mechanisms [39–41]. The variations in the cell cycleeffects of HSVtk/ganciclovir on different tumor cellof marked increase in in situ DNA fragment end-label-

ing (Fig. 2), and positive annexin V binding of phospha- types might be due to differences in the expression oftransduced HSVtk and cellular kinase activities, gan-tidylserine inversion on cell surface (Fig. 4). Further-

more, with the application of in situ DNA end-labeling ciclovir concentration, and incubation time. Presum-ably, with the DNA damages not properly repaired andmethod in tissue sections (Fig. 7), it is possible to dem-

onstrate in vivo the induction of apoptosis in association the cell cycle arrest abnormally prolonged, processes ofapoptosis would be activated [42]. This is consistentwith tumor regression of HSVtk-transduced B16F10

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74 WEI ET AL.

therapy for hepatoma cells using a retrovirus vector carryingwith our observation that it took at least 48 h of contin-herpes simplex virus thymidine kinase gene under the controluous ganciclovir exposure before the signs of apoptosisof human a-fetoprotein gene promoter. Cancer Res. 55, 3105–appeared in the HSVtk-expressing B16F10 melanoma 3109.

cells and that apoptosis was preceded first by sustained 8. Wills, K. N., Huang, W. M., Harris, M. P., Machemer, T., Ma-increase in p53 protein level and then cyclin B1 protein neval, D. C., and Gregory, R. J. (1995). Gene therapy for hepato-

cellular carcinoma: Chemosensitivity confered by adenovirus-level. Recently, Hamel et al. [36] reported that Bcl-2mediated transfer of the HSV-1 thymidine kinase gene. Cancerand perhaps other known apoptosis-inhibiting factors,Gene Ther. 3, 191–197.such as Bcl-XL, may influence the efficacy of HSVtk/

9. Culver, K. W., Ram, Z., Wallbridge, S., Ishii, H., Oldfield, E. H.,ganciclovir treatment, namely that tumors expressing and Blaese, R. M. (1992). In vivo gene transfer with retroviralhigh levels of Bcl-2 or Bcl-XL could be more resistant vector-producer cells for treatment of experimental brain tu-to this gene therapy strategy than tumors in which mors. Science 256, 1550–1552.apoptosis can be induced readily. We have, however, 10. Takamiya, Y., Short, M. P., Ezzeddine, Z. D., Moolten, F. L.,

Breakefield, X. O., and Martuza, R. L. (1992). Gene therapy offound no apparent changes in Bax or Bcl-2 protein levelmaligant brain tumors: A rat glioma line bearing the herpesprior to or during HSVtk/GCV-induced apoptosis in thesimplex virus type 1-thymidine kinase gene and wild type retro-B16F10 melanoma model. Instead, there was an in- virus kills other tumor cells. J. Neurosci. Res. 33, 493–503.

crease in the levels of Fas (APO-1/CD95) and its ligand 11. Barba, D., Hardin, J., Sadelain, M., and Gage, F. H. (1994).FasL that occurred kinetically between the cell cycle Development of anti-tumor immunity following thymidine ki-

nase-mediated killing of experimental brain tumors. Proc. Natl.arrest and apoptosis in B16F10 melanoma and otherAcad. Sci. USA 91, 4348–4352.three murine tumors subjected to the HSVtk/GCV

12. Rainov, N. G., Kramm, C. M., Guterman, K. A., Chase, M.,treatment (data to be published). In this regard, itUeki, K., Louis, D. N., Harsh, G. R. I. V., Chiocca, E. A., andwould be of interest to determine how the cell cycleBreakefield, X. O. (1996). Retrovirus-mediated gene therapy of

regulatory proteins-kinases and phosphatases may be experimental brain neoplasms using the herpes simplex virus-affected by the HSVtk/ganciclovir treament, leading thymidine kinase/ganciclovir paradigm. Cancer Gene Ther. 3,

99–106.eventually to apoptosis, and how combination with13. Freeman, S. M., McCune, C., Robinson, W., Abboud, C. N.,other apoptosis-causing chemotherapeutic agents may

Abraham, G. N., Angel, C., and Marrogi, A. (1995). The treat-be exploited to improve the therapeutic efficacy as wellment of ovarian cancer with a gene modified cancer vaccine: Aas to enhance the ‘‘bystander effect.’’ phase I study. Human Gene Ther. 6, 927–939.

14. Manome, Y., Abe, M., Hagen, M. F., Fine, H. A., and Kufe, D. W.We thank Dr. I. J. Fidler for kindly providing the original mouse (1994). Enhancer sequences of the DF3 gene regulate expres-

B16F10 melanoma cells. This work was supported by funding from sion of the herpes simplex virus thymidine kinase gene andDepartment of Health, R.O.C. ( DOH 84-TD-014) to NHRI, by an confer sensitivity of human breast cancer cells to ganciclovir.Academia Sinica cancer therapy program project grant, and by a Cancer Res. 54, 5408–5413.National Science Council grant (NSC 86-2314-B-075-051).

15. Plautz, G., Nabel, E. G., and Nabel, G. J. (1991). Selective elimi-nation of recombinant genes in vivo with a suicide retroviral

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Received November 5, 1997Revised version received January 6, 1998

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